Upon binding an antigen, an antibody can mobilize the immune system to clear—molecules bearing the antigen and to kill antigen-bearing organisms or cancer cells. Beyond that, the antibodies can mimic the effect of native ligands with their receptors, activating or blocking the signaling pathways of the receptors. Moreover, antibodies are capable of targeting nucleic acids, chemotherapeutic agents, toxins or radionuclides that are bound to the antibodies to the surface of cancer cells when the antibodies recognize an antigen that serves as a specific tumor marker, thereby eliminating non-specific damage caused by these agents to normal cells (Schrama D, et al. Nature Reviews of Drug Discovery 5: 147-159 (2006)). Indeed, in addition to the conjugation of chemotherapeutics, radionuclides or toxins, antibody-targeted “drugs” are being developed in many fields, including antibody-cytokine fusion proteins for immune modulation, antibody-ligand fusion protein for apoptosis induction, and antibody directed enzyme pro-drugs for drug delivery. For the intracellular delivery of conjugated molecules, the antibody must be endocytosed by the target cell since an internalized antibody conjugate can promote accumulation of the conjugated agents. Some linkers involved in such conjugation also need to be internalized to release the compounds by the lysosome Schrama et al, supra). Because epidermal growth factor (EGFR) receptors are overexpressed on solid tumors, EGFR is an ideal target for antibody-directed drug delivery. Preferred forms of antibodies for this purpose are human anti-EGFR antibodies or antigen-binding fragments thereof.
Since the 1970s, rodent, particularly murine, antibodies have been widely applied for medical purposes, mainly for in vitro diagnosis (Kohler G, et al. Nature 256:495-7 (1975)). The first clinical study with therapeutic murine monoclonal antibodies (mAbs) was performed in the early 1980s, but failed due to (a) induction of human anti-mouse antibodies (HAMA), (b) short serum half-lives, and (c) low efficacy of interaction with human immune effector cells (Reichert J M, et al. Nature Biotechnology 23:1073-8 (2005)). Human antibodies are desired for treatment and in vivo diagnosis of human subjects in order to prevent the potential immune response that would eliminate non-human antibodies and thereby decrease their effects after the first use. The development of recombinant technology has enabled the generation of a chimeric antibodies that combine the variable region of a mouse antibody and the constant region of a human antibody) or fully human antibodies for clinical use. Currently, three kinds of technologies are used to create human antibodies in quantities sufficient for clinical use. Fully human antibodies may be produced by a transgenic mouse, humanization modification of mice antibodies, or by selection (by panning) of recombinant human antibody libraries (Jakobovits, A. Curr Opin Biotechnol 6: 561-6 (1995); Jones P T, et al. Nature 321: 522-5 (1986)).
Fifty nine (59) genes in the human genome encode 20 distinct families of receptor tyrosine kinases that regulate a great diversity of cellular processes including cell survival, proliferation and differentiation (Manning G, et al. Science 298: 1912-34 (2002); Schlessinger J. Cell 103:211-25 (2002)). The 170-kDa epidermal growth factor (EGF) receptor, (also known as HER 1 or ErbB-1), one of the best studied tyrosine kinase receptors, has long been related to malignant diseases. EGFR is over-expressed in human solid tumors, including non-small cell lung cancer, prostate cancer, breast cancer, gastric cancer and tumors of the head and neck, promotes tumorigenesis, angiogenesis and metastasis, and in some cases is related to prognosis of malignant disease as well as patients' response to chemotherapy (Salomon D S, et al. Crit Rev Oncol Hematol 19: 183-232 (1995); Carbone D P. J Clin Oncol 21: 4268-69 (2003); Nicholson R I, et al. Eur J Cancer 37 (Suppl 4): S9-15) (2001)). Given the critical role of EGFR in the survival of cancer cells, and the overexpression of EGFR as a transmembrane protein located on the cell surface, it has long been considered to be a practical target for tumor immunotherapy (Holbro T, et al. Annu Rev Pharmacol Toxicol 44: 195-217 (2004)).
The mechanism by which antibodies produce therapeutic outcomes include antibody dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity, and the blocking of signal pathways that promote uncontrolled cell proliferation, e.g., with a neutralizing antibody (Von Mehren M, et al. Annu Rev Med 54:343-69 (2003)). In the case of the Erb family of EGFRs, antibodies have been developed to prevent heterodimerization of ErbB2 and ErbB3 (pertuzumab/Omnitarg™), to disrupt signal transduction (Trastuzumab/Herceptin™), and to prevent EGF binding to the EGFR (cetuximab/Erbitux™) (Molina M A, et al. Cancer Res 61:4744-49 (2001); Badache A, et al. Cancer Cell 5:299-301 (2004); Li S, et al. Cancer Cell 7:301-11 (2005)).
In addition, antibodies are good delivery vehicles for targeting agents to the EGFR for treatment or in vivo diagnosis. This can be done by conjugating chemotherapeutic drugs, radionuclides, or immunoliposomes for nanoscale delivery, and by directly fusing the antibody to an immunotoxin or cytokines using recombinant technology (Wu A M, et al. Nature Biotech 23:1137-46 (2005); Mamot C, et al. Cancer Res 65:11631-38 (2005)) Schrama D, et al., supra). An antibody-conjugated drug delivery system can direct toxic compounds to penetrate the targeted malignant tissue or cells specifically, thus decreasing the toxicity of drug to normal cells nearby and elsewhere, and increase the therapeutic effect by enhancing the accumulated dose of the drug in the targeted tissue. Conjugation also increases the solubility of some compounds in physiological solutions, enhancing the stability of the compounds, and preventing the conjugated drug from being pumped out of cells by the multidrug resistance associated p-glycoprotein transmembrane pump (Guillemard V, et al. Cancer Res 61: 694-9 (2001); Guillemard H, et al. Oncogene 23:3613-21 (2004)). Similarly, an antibody conjugated to a detectable label such as a radionuclide provides a method for early diagnosis of a malignancy, when a cell surface protein is overexpressed or mutated so that is distinguishable by an antibody from the unmutated form.
When using an antibody for drug delivery, internalization of the antibody is generally desired since it allows some compounds to be delivered intracellularly, for example after release, from the antibody. Schrama D, et al., supra
Relatively smaller antibody fragments have higher penetrating speed into the solid tumor tissue (Holliger P, et al. Nature Biotechnology 23: 1126-36 (2005)), plus, if they are xenogeneic to the host, they have fewer foreign epitopes that can be recognized by the recipient's immune system. To avoid such immunogenic effects of xenoantibodies, most antibodies in clinical trials or use are human antibodies or at least chimeric (and humanized) antibodies so that they comprise a human constant regions linked to the original, xenogeneic (typically murine) variable regions.
Antibody phage display technology provides ways to raise a human antibody (McCafferty J, et al. Nature 348:552-4 (1990)). This technology allows rapid isolation of antigen-binding antibody fragments through the use of “bio-panning” and bypassing the immunization and fusion procedure the use of which would be unethical in humans. The bio-panning procedure, permits harvesting of antibodies or fragments with desired function by different panning strategies. A useful antibody to be subjected to immunoconjugation should bind, for example, to a cell surface molecule in its native condition. In the process of seeking a functional antibody which binds to a native antigen in a cell having a specific physiological condition, selection of the antibody/fragment on cells of a cell line that typify that physiological condition would enhance the chances of success. Bio panning on living cells using particular protocols could increase the probability of selecting an internalizing antibody (Becerril B, et al. Biochem Biophys Res Comm 255:386-93 (1999)).